Fixation and Tissue Pretreatment
Lars-Inge Larsson in Immunocytochemistry: Theory and Practice, 2020
There are several aspects of fixation for immunocytochemistry. These include: Type of material and speciesType of fixativeRate of penetration and fixationFixative concentrationpHOsmolarityTemperatureType of treatment after fixation
Eosinophils in a Guinea Pig Model of Allergic Airways Disease
Gerald J. Gleich, A. Barry Kay in Eosinophils in Allergy and Inflammation, 2019
Only one of the monoclonal antibodies (designated 8A12) was found to be suitable for use in immunocytochemistry. This stained eosinophils strongly and specifically in cytospin preparations, frozen sections, or wax-embedded sections (after protease digestion). Either indirect immunofluorescence or alkaline phosphatase–anti-alkaline phosphatase techniques could be used to visualize the staining. This resulted in strong granular staining of the eosinophils and proved to be an extremely sensitive means of localizing this cell type in tissue sections. The 8A12 monoclonal was also capable of localizing MBP deposited extracellularly by eosinophils at sites of allergic inflammation. Staining with this antibody was specific for eosinophils and did not stain other guinea pig leukocyte types.
Monoclonal Antibodies in Diagnosis, Prognosis, and Therapy of Human Carcinomas: Clinical Impact 20 Years Later
Maurizio Zanetti, J. Donald Capra in The Antibodies, 1999
For the detection of tissue antigen, immunohistochemistry and immunocytochemistry are the most frequently used methodologies for diagnostic and prognostic purposes. Radiolabeled antibodies tested on tissues sections and detected by autoradiography are also used, especially when an estimate of the affinity of the antigen-antibody reaction is desired, as in the case of selection of candidate patients for immunotherapy. Immunobiochemical methods performed on tissue extracts offer the advantage of giving quantitative results, although these may be misleading when dealing with heterogeneous material such as extracts of tumors with poor cellularity or extensive lymphoid infiltration. The requirement for a consistent amount of tissue to be solubilized, the lack of control of the sample quality, and the cost of this test have led to the search for alternative methodologies. Immunohistochemistry (Figure 2) offers many advantages, including reduced quantity of material, low cost, rapid execution, and feasibility in all laboratories. On the other hand, lack of standardization among different laboratories has led to difficulties in comparing results [14].
RhoA Activation Decreases Phagocytosis of Trabecular Meshwork Cells
Published in Current Eye Research, 2021
Tomokazu Fujimoto, Saori Sato-Ohira, Hidenobu Tanihara, Toshihiro Inoue
TM cells were cultured on gelatin-coated glass cover slips for immunocytochemistry. Immunocytochemistry was conducted as described previously.25 The cells were fixed with 4% (v/v) paraformaldehyde in PBS for 15 min at room temperature, and then washed with cytoskeletal buffer (10 mM 2-morpholinoethansulfonic acid potassium salt, 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, and 5 mM glucose, pH 6.1). For permeabilization, cells were treated with 0.5% (v/v) Triton X-100 in PBS for 12 min at room temperature. The cells were then blocked with serum buffer (10% FBS and 0.2 mg/mL sodium azide in PBS) at 4°C for at least 2 h and were then treated with anti-vinculin, anti-YAP, or anti-TAZ antibodies at 4°C overnight. Afterwards, the cells were incubated with the anti-mouse IgG secondary antibody Alexa Fluor 488, and Alexa Fluor 546 phalloidin at room temperature for 30 min. After the cells had been mounted with VECTASHIELD mounting medium containing 4ʹ, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA), they were observed under an all-in-one epifluorescence microscope (BZ-X710; Keyence). We performed immunostaining in at least three independent experiments.
The Texas Society of Pathologists: molded by the legacy of pathology and focused on excellence in medicine for 100 years and beyond
Published in Baylor University Medical Center Proceedings, 2021
Pathology has always been an opportunistic and eclectic science, taking advantage of advances in basic sciences to conduct basic and translational research ultimately aimed at the elucidation of the etiology and pathogenesis of human diseases. In the 18th and 19th centuries, autopsy pathology was primarily responsible for the scientific elucidation of many human diseases. In the 20th and 21st centuries, autopsy pathology has continued to be primarily responsible for the discovery or elucidation of the pathogenesis of new diseases, such as acquired immunodeficiency syndrome due to human immunodeficiency virus, as well as documentation of effects of new therapies.138–141 Both the discovery process as well as diagnostic pathology have been enhanced by the coupling of gross examination and light microscopy with new techniques, including electron microscopy, fluorescence microscopy, histochemistry, and immunohistochemistry. From the 1970s onward, immunocytochemistry has become a powerful and ubiquitous component of diagnostic pathology.142
Differentiation and Maturation Effect of All-Trans Retinoic Acid on Cultured Fetal RPE and Stem Cell-Derived RPE Cells for Cell-Based Therapy
Published in Current Eye Research, 2022
Tingyu Yan, Na Yang, Wei Hu, Xinxin Zhang, Xuedong Li, Youjin Wang, Jun Kong
P1 and P5 fRPE cultured for 1 month were compared. Using RT-qPCR analysis, we assayed the mRNA expression levels of RPE markers including RPE65, ZO-1, E-cadherin, and mesenchymal markers including α-SMA, FN, N-cadherin. We found that expression of RPE65, E-cadherin, and ZO-1 in fRPE cells that underwent EMT changes were significantly decreased and expression of a-SMA, N-cadherin, and FN were significantly increased by 4.12-folds, 3.21-folds, and 10.26-folds, respectively (p < 0.05) (Figure 3(a)). In a parallel approach, western blotting results demonstrated that the protein expression of RPE markers was downregulated and the protein expression of EMT markers was upregulated in EMT-fRPE cells (Figure 3(b)). In addition, immunocytochemistry results strengthened the findings (Figures 3(c,d)).
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